The aims of the proposed research are to study the interaction of tubulin with specific, tight-binding ligands and apply the information obtained to elucidating the properties of tubulin. Tubulin is present in eucaryotic organisms and can exist in a polymeric form called microtubules. Microtubules have a role in several diverse cellular functions, e.g., mitosis, secretion, hormonal action, but in the presence of ligands such as colchicine these processes are disrupted. The ultimate goals of the proposed research are elucidation of the conformational changes occurring on ligand binding to tubulin and isolation of the suspected endogenous inhibitor/regulator of microtubule formation. Examination of the colchicine binding site through fluorescent and spin-labeled analogs will provide the necessary data to achieve these goals. Deacetylcolchicine (DAC) and [4-3H]-DAC allow the synthesis of a variety of ligands that show tight binding to tubulin. The fluorescent derivatives provide information on the microenvironment and binding characteristics of tubulin through fluorescence emission spectroscopy and polarization measurements. Equilibrium dialysis and an HPLC modification of the Hummel-Dreyer procedure to determine equilibrium binding parameters will give additional information on the nature of the CLC or analog binding to tubulin. The spin-labeled derivatives examine the binding site by EPR, circular dichroism (CD) and magnetic CD as well as probe conformational changes that may occur in tubulin. Knowledge of the binding site, preparation of a tubulin-affinity resin and preparation of a colchicine-affinity resin will allow purification of tubulin as well as isolation of the endogenous ligand. The ligand interactions between tubulin and colchicine analogs will provide fundamental information regarding tubulin structure. The research proposed involves techniques of synthetic, bioorganic, biophysical, and biochemical protein chemistry.